Interpretive Summary: Huanglongbing (HLB), also known as citrus greening disease, is currently the greatest threat to the citrus industry worldwide. It is caused by an unusual bacterium that lives inside the cells of the sweet orange trees and its insect vector. There is no cost-effective control for this disease. The bacterium, called ‘Liberibacter’ is related to a large group of beneficial bacteria, but has experienced an evolutionary process that has led to a genome that has an unusual ratio of the ‘letters’ that make up the genetic code. What are the consequences of this unusual genetic code? We have studied the genome of ‘Liberibacter’ in detail by comparing it gene for gene with other related bacteria to find out if there are other unusual features of the genome. In this process we hope to find features of the genome that can be exploited to better control this bacterium and the citrus greening disease. We have found for example that ‘Liberibacter’ has the complete gene set required to copy its genome. However it lacks the genes necessary to make the genetic ‘letters’ ‘from scratch’, and so must obtain these basic components for life from the host cells. Mistakes in copying the DNA code are inevitable, but ‘Liberibacter’ seems to be relatively unable to repair such mistakes by the processes that other bacteria use, because it does not have many of the enzymes necessary for this task. This suggests that errors in the genetic code will accumulate. ‘Liberibacter’ lives inside plant or insect cells, which is a very sheltered environment, free of many stresses present in the larger world. Perhaps as a consequence, ‘Liberibacter’ lacks multiple layers of genetic control mechanisms necessary to deal with stresses in the larger world. Thus ‘Liberibacter’ can’t make the ‘letters’ of the genetic code itself but must import them; it has difficulty in correcting routine genetic mistakes; and it has a reduced and apparently inflexible program for controlling gene expression. Perhaps these apparent weaknesses can be exploited to control this pathogen. Our results will be of interests to scientists who seek to better understand the biochemistry and genetics of this pathogen in order to develop methods to control it

Technical Abstract:
An intracellular plant pathogen ‘Ca. Liberibacter asiaticus,’ a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, Agrobacterium tumefaciens and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 52 clusters of conserved orthologous genes found on the chromosomes of all five metabolically diverse species. The intracellular pathogens, ‘Ca. Liberibacter asiaticus’ and Bartonella henselae have genomes drastically reduced gene content and size as well as a relatively low content of guanine and cytosine. Codon and amino acid preferences that emphasize low guanosine and cytosine usage are globally employed in these genomes, including within regions of microsynteny and within signature sequences of orthologous proteins. The length of orthologous proteins is generally conserved, but not their isoelectric points, consistent with extensive amino acid substitutions to accommodate selection for low GC content. The ‘Ca. Liberibacter asiaticus’ genome apparently has all of the genes required for DNA replication present in Sinorhizobium meliloti except it has only two, rather than three RNaseH genes. The gene set required for DNA repair has only one rather than ten DNA ligases found in Sinorhizobium meliloti, and the DNA PolI of ‘Ca. Liberibacter asiaticus’ lacks domains needed for excision repair. Thus the ability of ‘Ca. Liberibacter asiaticus’ to repair mutations in its genome may be impaired. Both ‘Ca. Liberibacter asiaticus and Bartonella henselae lack enzymes involved with the metabolism of purines and pyrimidines, which must therefore be obtained from the host. The ‘Ca. Liberibacter asiaticus’ genome also has a greatly reduced set of sigma factors used to control transcription, and lacks sigma factors 24, 28 and 38. The ‘Ca. Liberibacter asiaticus’ genome has all of the hallmarks of a reduced genome of a pathogen adapted to an intracellular lifestyle. We hypothesize that replication and transcription of an A+T rich genome is energetically favored by a lower ATP cost for strand separation by DNA helicase. The A+T rich genomes of obligate or semi-obligate intracellular pathogens may be positively selected for by energy savings for both the pathogen and the host.